We propose to study the development of drug resistance in the context of HIV protease inhibition to develop and test structural and synthetic strategies in response to this mechanism. The overall goal of this Program Project is to understand the mechanisms of viral resistance, enabling modeling and design of more sustainable anti-viral therapeutic strategies. The Program consist of four highly integrated Projects and three supporting Core facilities: Project 1, will enhance and extend a computational co-evolution approach to drug resistance by developing and applying detailed atomic models of drug/target interactions, modeling viral population dynamics and patient response under drug selection pressure, exploiting automated learning and hidden Markov modeling approaches to inform and refine these models; Project 2 will exploit the capabilities of high throughput crystallography to find and characterize novel binding sites on the protein target using fragment libraries to help construct new inhibitor leads to maintain efficacy against multi-site PR mutants, linking with optimization and synthetic efforts in Projects 1 and 3 respectively; Project 3 will utilize their Click Chemistry synthetic approaches for rapid development and evolution of novel fragment-based inhibitors in conjunction with Projects 1 and 2, and develop resistance probes with Project 4; Project 4 will experimentally characterize the evolution of HIV resistance in response to protease inhibition both within PR and in the rest of Gag-Pol, by exploiting tissue-culture time-course evaluation passaged virus in the presence of identified inhibitors, as well as from deep genetic analysis of selected patient samples; Core A will provide mutant and wildtype proteases, functional assays and chemical probes, and inhibitor analyses for the Program; Core B will provide the necessary x-ray structural data and computational analysis to integrate new information on protease mutants, and protease-inhibitor interactions; and Core C will assemble and make available to projects 1 and 4 time-course anti-retroviral treatment data on HIV infected patients as well as blood samples from highly resistant individuals for in-depth bioinformatic and viral genomic analyses. AIDS remains the major pandemic of our time. While patients infected with HIV can now be treated with drugs that enable them to live productive lives, the virus can subvert this treatment by developing resistance to these drugs. This study is aimed at a detailed understanding of HIV drug resistance, with the goal of developing new therapeutic strategies for more sustainable treatments to prevent AIDS.